Netrins

ABSTRACT

Netrin proteins, nucleic acids which encode netrin proteins and hybridization reagents, probes and primers capable of hybridizing with netrin genes and methods for screening chemical libraries for lead compounds for pharmacological agents are provided.

RELATED APPLICATIONS

This is application is a continuation of Ser. No. 08/482,677, filed Jun. 7, 1995, now U.S. Pat. No. 6,017,714 which is a continuation in-part-of Ser. No. 08/152,019, filed Nov. 12, 1993, now U.S. Pat. No. 5,565,331, both of which are incorporated herein by reference

The research carried out in the subject application was supported in part by grants from the National Institutes of Health. The government may have rights in any patent issuing on this application.

INTRODUCTION

1. Field of the Invention

The field of this invention is vertebrate netrin proteins and genes which are involved in neural axon outgrowth.

2. Background

In the developing nervous system, axons project considerable distances along stereotyped pathways to reach their targets. Axon growth and guidance depends partly on the recognition of cell-surface and extracellular matrix cues along these pathways. The identification of such nerve cell growth and guidance cues is the holy grail of neurobiology. These are the compounds that tell neurons when to grow, where to grow, and when to stop growing. The medical applications of such compounds are enormous and include modulating neuronal growth regenerative capacity, treating neurodegenerative disease, and mapping (e.g. diagnosing) genetic neurological defects.

Over decades of concentrated research, various hypotheses involving chemo-attractants and repellents, labeled pathways, cell adhesion molecules, etc. have been invoked to explain guidance. Molecules such as N-CAM and N-cadherin have been reported to provide favorable substrates for axon growth and certain sensory axons may be responsive to NGF and NGF-like factors. Recent reports suggest the existence of diffusible chemotropic molecule(s) which influence the pattern and orientation of commissural axon growth.

Relevant Literature

Placzek et al. (1990) Development 110, 19-30; Placzek et al. (1990) Cold Spring Harbor Symposia on Quantitative Biology 55, 279-302.; and Tessier-Lavigne et al. (1988) Nature 336: 775-778 report evidence for diffusible chemotropic molecules which influence the pattern and orientation of commissural axon growth. Gundersen and Barret (1980) JCB 87, 546-554, Lohof et al. (1992) J. Neurosci. 12 (4), 1253-1261 and Zheng et al. (1993) Soc. Neurosci. Abstr 19, 608.9 report neural chemotaxis in response to NGF, cAMP and acetylcholine, respectively. Ishii et al. (1992) Neuron 9, 873-881 disclose a gene, unc-6, derived from C. elegans, which has sequence similarity to the disclosed netrins. Data disclosed in this application was published in Serafini et al (1994) Cell 78, 409-424 and Kennedy et al (1994) Cell 78, 425-435 at page 5, column 1. The work was also reported in The New York Times, Section B7, Tuesday, Aug. 16, 1994 and more recently (May 19, 1995) described in Science 268, 971-973 (see also references cited therein).

SUMMARY OF THE INVENTION

The invention provides methods and compositions relating to netrins and netrin genes. Netrins are a novel class of proteins which are naturally involved in neural axon guidance. The subject compositions include nucleic acids which encode netrin proteins and hybridization probes and primers capable of hybridizing with netrin genes. Netrins find particular use in modulating neural axon outgrowth. The disclosed compositions also find use variously in screening chemical libraries for regulators of axon outgrowth and orientation, in genetic mapping, as probes for related genes, as diagnostic reagents for genetic neurological disease and in the production of specific cellular and animal systems for the development of neurological disease therapy.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides methods and compositions relating to netrins and netrin genes; including methods and compositions for identifying, purifying, characterizing, and producing netrins and for identifying, characterizing, cloning, expressing, inhibiting the expression of and amplifying netrin genes.

Netrins are characterized by sequence similarity to the disclosed netrins 1 and 2. Using the amino acid sequence search program BLASTP (Altschul et al. (1990) Basic Local Alignment Search Tool, J Mol Biol 215, 403-410), complete (full length) netrin amino acid sequences provide a Probability P(N) score of less than 1.0e⁻²⁰⁰. In contrast, complete amino acid sequence comparison of a netrin with the evolutionarily related laminin proteins provides P(N) scores exceeding 1.0e⁻¹⁴⁴. In addition, netrins generally show at least about 25% overall pair-wise sequence identity with all of the disclosed netrins 1 and 2 and at least about 50% pair-wise sequence identity within domain V. Furthermore, netrins are generally characterized by netrin-specific amino acid sequences invariant across the disclosed netrins 1 and 2 as seen in their amino acid alignments. The subject netrins may be incomplete translates of the disclosed netrin cDNA sequences or deletion mutants of the corresponding conceptual translates, which translates or deletion mutants have the netrin binding activity and specificity described herein.

Netrin peptides of the invention comprise unique portions of the disclosed netrin polypeptides and netrin receptors. A “unique portion” has an amino acid sequence unique to that disclosed in that it is not found in any previously known protein and has a length at least long enough to define a novel peptide. Unique portions are found to vary from about 5 to about 25 residues, preferably from 5 to 10 residues in length, depending on the particular amino acid sequence and are readily identified by comparing the subject portion sequences with known peptide/protein sequence data bases. Preferred unique portions include netrin residues that directly bind and activate (agonize) netrin receptors, especially residues that derive from the EGF-like domains of the disclosed sequences, especially those of the human varieties.

Particular preferred netrin peptides are listed here. These peptides are shown by functional assays disclosed herein to have biological activity including axon outgrowth and/or orienting activity. It is apparent to those of ordinary skill in the art that substitutions of chemically conservative residues can be made while preserving function.

Preferred peptides derived from domain V of netrin 2 and netrin 1:

1. NGH AA/SR (SEQ ID NO:01/02, residues 289-294/265-270)

2. VRD RDD N/SLV (SEQ ID NO:01, residues 296-304)

3. VKD KEQ KLV (SEQ ID NO:02, residues 272-280)

4. KHN TE/AG PE (SEQ ID NO:01/02, residues 308-315/284-291)

5. KPF HYD DRP WQR AT/SA REA NE (SEQ ID NO:01/02, residues 320-338/296-319)

6. NLH ARR (SEQ ID NO:01, residues 345-350)

7. RFN MEL YKL SGR KSG GV (SEQ ID NO:01/02, residues 352-368/328-344)

8. RHN TAG RH (SEQ ID NO:01/02, residues 373-380/349-356)

9. KEG FYR DLS KP/SIS/ TH/DR KA (SEQ ID NO:01/02, residues 385-401/361-377)

10. HPV GAA GK/QT (SEQ ID NO:01/02, residues 408-416/384-392)

11. NQT TGQ (SEQ ID NO:01/02, residues 418-423/394-399)

12. KDG VTG I/LT (SEQ ID NO:01/02, residues 427-434/403-410)

13. AKG Y/FQQ SRS PI/VA P (SEQ ID NO:01/02, residues 439-451/415-427)

Preferred peptides derived from the C terminal domains of netrin 2 and netrin 1:

14. IKI PAI/AN/P (SEQ ID NO:01/02, residues 453-459/429-435)

16. STE A/EPA DCD SYC K (SEQ ID NO:01/02, residues 466-478/442-454)

17. KI/MN MKK YCK/R KDY V/AVQ (SEQ ID NO:01/02, residues 485-499/461-475)

18. KFT I/VNI L/T/ISV YK (SEQ ID NO:01/02, residues 513-523/489-499)

19. CKC PKI/V (SEQ ID NO:01/02, residues 545-550/521-526)

20. ADK S/NSL VIQ WRD (SEQ ID NO:01/02, residues 573-584/549-560)

21. RLR RGD QTL W (SEQ ID NO:01, residues 528-537)

22. RVK RGD NFL W (SEQ ID NO:02, residues 504-513)

Preferred peptides derived from domain VI of netrin 2 and netrin 1:

23. DPC YDE (SEQ ID NO:01/02, residues 40-45/27-30)

24. RCI PE/DF VNA/S AFG KEV (SEQ ID NO:01/02, residues 51-65/38-52)

25. SST CGK PP (SEQ ID NO:01/02, residues 68-75/55-62)

26. A/SSD PKR/K AHP PA/S (SEQ ID NO:01, residues 97-107)

27. LTD LNN PH (SEQ ID NO:01, residues 109-116)

28. LTD LNT AA (SEQ ID NO:02, residues 80-87)

29. NL/MT CWR/Q S—(SEQ ID NO:01/02, residues 117-123/88-94)

The claimed netrins are isolated, partially pure or pure and are typically recombinantly produced. An “isolated” protein for example, is unaccompanied by at least some of the material with which it is associated in its natural state and constitutes at least about 0.5%, preferably at least about 2%, and more preferably at least about 5% by weight of the total protein in a given sample; a partially pure protein constitutes at least about 10%, preferably at least about 30%, and more preferably at least about 60% by weight of the total protein in a given sample; and a pure protein constitutes at least about 70%, preferably at least about 90%, and more preferably at least about 95% by weight of the total protein in a given sample. A wide variety of molecular and biochemical methods are available for generating and expressing the subject compositions, see e.g. Molecular Cloning, A Laboratory Manual (Sambrook, et al. Cold Spring Harbor Laboratory), Current Protocols in Molecular Biology (Eds. Aufubel, et al., Greene Publ. Assoc., Wiley-Interscience, N.Y.) or that are otherwise known in the art. The disclosed netrin peptides are also used as immunogens to generate specific polyclonal or monoclonal antibodies. See, Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, for general methods.

The disclosed netrin compositions may be used to modulate axon outgrowth or guidance in situ or in vivo. For in vivo applications, the compositions are added to a retained physiological fluid such as blood or synovial fluid. For CNS administration, a variety of techniques are available for promoting transfer of the therapeutic across the blood brain barrier including disruption by surgery or injection, drugs which transiently open adhesion contact between CNS vasculature endothelial cells, and compounds which facilitate translocation through such cells. Netrins may also be amenable to direct injection or infusion, topical, intratracheal/nasal administration e.g. through aerosol, intraocularly, or within/on implants e.g. fibers e.g. collagen, osmotic pumps, grafts comprising appropriately transformed cells, etc. A particular method of administration involves coating, imbedding or derivatizing fibers, such as collagen fibers, protein polymers, etc. with therapeutic proteins. Other useful approaches are described in Otto et al. (1989) J Neuroscience Research 22, 83-91 and Otto and Unsicker (1990) J Neuroscience 10, 1912-1921. Generally, the amount administered will be empirically determined, typically in the range of about 10 to 1000 μg/kg of the recipient and the concentration will generally be in the range of about 50 to 500 μg/ml in the dose administered. Other additives may be included, such as stabilizers, bactericides, etc. will be present in conventional amounts.

The invention provides netrin-specific binding agents including isolated binding targets such as membrane-bound netrin receptors and netrin-specific antibodies and binding agents identified in screens of natural and synthetic chemical libraries, and methods of identifying and making such agents, and their use in diagnosis, therapy and pharmaceutical development. Generally, netrin-specificity of the binding agent is shown by binding equilibrium constants. Such agents are capable of selectively binding a netrin, i.e. with an equilibrium constant at least about 10⁷M⁻¹, preferably at least about 10⁸M⁻¹, more preferably at least about 10⁹M⁻¹. A wide variety of cell-based and cell-free assays may be used to demonstrate netrin-specific binding; preferred are rapid in vitro, cell-free assays such as mediating or inhibiting netrin-cell/protein binding, immunoassays, etc.

The invention also provides nucleic acids encoding the subject proteins, which nucleic acids may be part of netrin-expression vectors and may be incorporated into recombinant cells for expression and screening, transgenic animals for functional studies (e.g. the efficacy of candidate drugs for neural disease or injury), etc. and nucleic acid hybridization probes and replication/amplification primers having a netrin cDNA specific sequence. The hybridization probes contain a sequence common or complementary to the corresponding netrin gene sufficient to make the probe capable of specifically hybridizing to the corresponding netrin gene in the presence of laminin genes. Hybridization probes having in excess of 100 continuous bases of netrin gene sequence are generally capable of hybridizing to the corresponding netrin cDNA and remaining bound at a reduced final wash stringency of 0.2×SSC (0.9 M saline/0.09 M sodium citrate) and 0.1% SDS buffer at a temperature of 65° C.

Netrin genes, the term including natural genomic and mRNA/cDNA sequences, are characterized by sequence similarity to the disclosed netrin 1 and 2 cDNAs. Using the nucleic acid sequence search program BLASTX (Altschul et al. (1990) Basic Local Alignment Search Tool, J Mol Biol 215, 403-410), complete coding region (full length) netrin cDNA sequences provide a Probability P(N) score of less than 1.0e⁻²⁰⁰. In contrast, complete coding region nucleic acid sequence comparison of a netrin cDNA with the evolutionarily related laminin cDNAs provides P(N) scores exceeding 1.0e⁻¹⁴⁴. In addition, netrin cDNAs generally show at least about 25% overall coding region pair-wise sequence identity with the disclosed netrins 1 and 2 cDNAs and at least about 35% domain V coding region pair-wise sequence identity. Furthermore, netrin genes are generally characterized by netrin gene-specific nucleic acid sequences invariant across the disclosed netrin 1 and 2 cDNAs as seen in their nucleic acid alignments. Vertebrate netrin genes derive from vertebrates.

The subject nucleic acids are isolated, meaning they comprise a sequence joined to a nucleotide other than that which it is joined to on a natural chromosome and usually constitutes at least about 0.5% , preferably at least about 2%, and more preferably at least about 5% by weight of total nucleic acid present in a given fraction. A partially pure nucleic acid constitutes at least about 10%, preferably at least about 30%, and more preferably at least about 60% by weight of total nucleic acid present in a given fraction. A pure nucleic acid constitutes at least about 80%, preferably at least about 90%, and more preferably at least about 95% by weight of total nucleic acid present in a given fraction. The subject nucleic acids find a wide variety of applications including use as translatable transcripts, hybridization probes, PCR primers, therapeutic nucleic acids, etc.; use in detecting the presence of netrin genes and gene transcripts, in detecting or amplifying nucleic acids encoding other netrins, and in gene therapy applications, e.g. antisense oligonucleotides capable of inhibiting the intracellular expression of a targeted netrin transcript.

The invention provides efficient methods of identifying pharmacological agents or lead compounds for agents capable of mimicking or modulating netrin function (e.g. bioactive netrin deletion mutants and netrin peptides). A wide variety of screens may be used; for example, cell-based assays for may be used for monitoring netrin function and in vitro binding assays may be used to identify netrin-specific binding agents. Tessier-Lavigne et al. (1988, supra) describe an assay for netrin activity and Kennedy et al. (1994) Cell 78, 425-435 describe a particularly convenient COS cell-based netrin expression assay. Preferred methods are amenable to automated, cost-effective high throughput screening of natural and synthetic chemical libraries for lead compounds. Identified reagents find use in the pharmaceutical industries for animal and human trials; for example, the reagents may be derivatized and rescreened in in vitro and in vivo assays to optimize activity and minimize toxicity for pharmaceutical development.

Netrin Gene Cloning Strategies

Vertebrate netrin genes are cloned using the using the two general cloning strategies illustrated below for mouse and human netrins. First, using a strategy based on the initial amplification of a PCR product, oligonucleotide primers are designed using amino acid and nucleic acid sequences conserved among the previously identified vertebrate netrin sequences. Using these primers, a partial cDNA clone,corresponding to the novel netrin of interest is amplified from cDNA ergonomic DNA from the tissue and organism of interest by PCR. This partial clone is then used to generate a labeled probe which is used to screen a cDNA library or genomic library at high stringency to isolate a full length cDNA corresponding to the clone of interest. We describe below how such a strategy, based on PCR followed by library screening, has been used to successfully isolate mouse netrin-1, Drosophila netrin-a, and two human netrin cDNAs. The second general strategy utilizes reduced stringency library screening (Sambrook et al., 1989). We demonstrate below the applicability of this method in the isolation of mouse netrin-2. In this case we amplified and incorporated ³²P into a probe which corresponded to domains VI and V in chicken netrin-2. Domains VI and V contain a number of regions of sequence which are well conserved among all vertebrate netrin family members isolated to this date. This probe was then used to screen an embryonic mouse brain cDNA library at reduced stringency. Our cloning of mouse netrin-2 using this method demonstrates that hybridization conditions are conveniently established which will detect netrin sequences between vertebrate species while avoiding significant background hybridization to non-netrin clones.

Our data identify netrin sequences common to the vertebrate netrins, mouse netrin-1, chicken netrin-1, and chicken netrin-2, which are not shared by the invertebrate netrin unc-6 as seen in netrin sequence alignments. The presence of these sequences, specific to vertebrates and conserved in all vertebrate netrins isolated, provides the necessary and sufficient sequence informative for generating primers and/or probes for any vertebrate netrin gene. In addition, amino acid sequence alignments similarly demonstrate that the vertebrate netrins define a structural class sharing common sequences not shared-with the invertebrate species illustrated by C. elegans unc-6 and the Drosophila Melanogaster netrin-a. Furthermore the alignment between the Drosophila and the C, elegans sequences indicates there is a greater diversity of netrin amino acid sequence represented within the invertebrate phylum than is present within the sequences derived from the vertebrate phylum.

The following examples are offered by way of illustration and not by way of limitation.

EXAMPLES

We isolated chicken netrin 1 and 2 cDNAs as described in Serafini et al. (1994) Cell 78:409-424. Based on the ckick netrin 1 and 2 cDNA sequences, we designed degenerate oligonucleotide primers and used these primers to amplify a cDNA encoding mouse netrin 1 from a murine cDNA library.

We isolated a mouse netrin-2 cDNA from a screen of a P0 (day of birth) mouse brain cDNA library (stratagene 937319: mouse P0 brain cDNA library in ZAPXP, oriented cloning). The probe used corresponded to sequences within domains VI and V of chicken netrin-2. Domain VI and V of the netrins contain regions of nucleic acid and amino acid sequence highly conserved in all netrins in each of the vertebrate species characterized to this date. The probe was labeled by incorporation of ³²P during PCR using a template of chicken netrin-2 cDNA.

1×10⁶ clones were screened at a reduced final wash stringency of 0.2×SSC and 0.1% SDS at 65 A1C (Sambrook et al., 1989). A single ˜7.5 kb clone corresponding to mouse netrin-2 cDNA was obtained. Sequencing of this cDNA indicated that it comprises over 40% of the netrin coding sequence but is lacking sequence corresponding to the 5′ coding sequence of mouse netrin-2. To isolate the 5′ coding sequence from cDNA isolated from cDNA libraries, we have employed a combination of library screening and PCR using standard methods (Sambrook et al., 1989).

We first isolated a human netrin cDNA using the degenerate oligonucleotide primers. The primers were constructed using amino acid sequences conserved in the previously isolated chick and mouse netrin sequences as a guide. The starting material for PCR was 100 ng of human genomic DNA. PCR products were subcloned and individual clones containing inserts corresponding to human netrin sequence isolated using a Grunstein and Hogness screen (Sambrook, 1989). ³²P was incorporated into a probe using PCR with a portion of the mouse netrin-1 cDNA clone as a template. The final wash of the filters was at a reduced stringency of 1×SSC and 0.1% SDS at 65° C. (Sambrook et al., 1989). This screen isolated an approximately 140 base pair human netrin cDNA clone. This cDNA fragment was used to isolate a longer human netrin cDNA from a Human fetal brain cDNA library (Stratagene cat#936206). The ˜140 base pair human netrin cDNA was used as a template and 32P incorporated into a human netrin cDNA probe using PCR. 1×10⁶ clones were screened at high stringency (Sambrook et al., 1989)identifying a single approximately 7 kb netrin cDNA. Sequence obtaining from the ends of this clone encode untranslated DNA sequence(see tables 9 and 10), indicating a full length clone. Internal sequence of the cDNA obtained using oligonucleotide primers corresponding to sequences contained in the ˜140 clone, confirm and extend that sequence in the larger clone. Searches of the NBRF amino acid and nucleic acid sequence databases indicate that the published sequences with which this human cDNA shares the highest sequence identity are those of chicken netrin-1 and chicken netrin-2, the only vertebrate netrin sequences contained in the database at this date. In addition, these sequences encode amino acid sequences indicating that this clone represents a human netrin cDNA.

A partial cDNA was first amplified by PCR using non-degenerate primers designed using the codon usage for Drosophila as a guide. The particular sequences used were chosen on the basis of their conservation in the amino acid sequences of the invertebrate netrin gene unc-6 and the chicken netrin-1 and netrin-2 cDNAs. Nested PCR amplification was performed using 1 ng of total embryonic Drosophila cDNA as a template. A full length cDNA corresponding to Drosophila melanogaster netrin-a was then isolated by screening a cDNA library at high stringency using standard methods ( Sambrook et al., 1989).

All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

4 605 amino acids amino acid single linear peptide not provided 1 Met Pro Arg Arg Gly Ala Glu Gly Pro Leu Ala Leu Leu Leu Ala Ala 1 5 10 15 Ala Trp Leu Ala Gln Pro Leu Arg Gly Gly Tyr Pro Xaa Leu Asn Met 20 25 30 Phe Ala Val Gln Thr Xaa Ala Asp Pro Cys Tyr Asp Glu His Gly Leu 35 40 45 Pro Xaa Arg Cys Ile Pro Asp Phe Val Asn Ser Ala Phe Gly Lys Glu 50 55 60 Val Lys Val Ser Ser Thr Cys Gly Lys Pro Pro Ser Arg Tyr Cys Val 65 70 75 80 Val Thr Glu Lys Gly Glu Glu Gln Val Arg Ser Cys His Leu Cys Asn 85 90 95 Ala Ser Asp Pro Lys Arg Ala His Pro Pro Ser Phe Leu Thr Asp Leu 100 105 110 Asn Asn Pro His Asn Leu Thr Cys Trp Gln Ser Asp Ser Tyr Val Gln 115 120 125 Tyr Pro His Asn Val Thr Leu Thr Leu Ser Leu Gly Lys Lys Phe Glu 130 135 140 Val Thr Tyr Val Ser Leu Gln Phe Cys Ser Pro Arg Pro Glu Ser Met 145 150 155 160 Ala Ile Tyr Lys Ser Met Asp Tyr Gly Lys Thr Trp Val Pro Phe Gln 165 170 175 Phe Tyr Ser Thr Gln Cys Arg Lys Met Tyr Asn Lys Pro Ser Arg Ala 180 185 190 Ala Ile Thr Lys Gln Asn Glu Gln Glu Ala Ile Cys Thr Asp Ser His 195 200 205 Thr Asp Val Arg Pro Leu Ser Gly Gly Leu Ile Ala Phe Ser Thr Leu 210 215 220 Asp Gly Arg Pro Thr Ala His Asp Phe Asp Asn Ser Pro Val Leu Gln 225 230 235 240 Asp Trp Val Thr Ala Thr Asp Ile Lys Val Thr Phe Ser Arg Leu His 245 250 255 Thr Phe Gly Asp Glu Asn Glu Asp Asp Ser Glu Leu Ala Arg Asp Ser 260 265 270 Tyr Phe Tyr Ala Val Ser Asp Leu Gln Val Gly Gly Arg Cys Lys Cys 275 280 285 Asn Gly His Ala Ser Arg Cys Val Arg Asp Arg Asp Asp Asn Leu Val 290 295 300 Cys Asp Cys Lys His Asn Thr Ala Gly Pro Glu Cys Asp Arg Cys Lys 305 310 315 320 Pro Phe His Tyr Asp Arg Pro Trp Gln Arg Ala Thr Ala Arg Glu Ala 325 330 335 Asn Glu Cys Val Ala Cys Asn Cys Asn Leu His Ala Arg Arg Cys Arg 340 345 350 Phe Asn Met Glu Leu Tyr Lys Leu Ser Gly Arg Lys Ser Gly Gly Val 355 360 365 Cys Leu Asn Cys Arg His Asn Thr Ala Gly Arg His Cys His Tyr Cys 370 375 380 Lys Glu Gly Phe Tyr Arg Asp Leu Ser Lys Pro Ile Ser His Arg Lys 385 390 395 400 Ala Cys Lys Glu Cys Asp Cys His Pro Val Gly Ala Ala Gly Gln Thr 405 410 415 Cys Asn Gln Thr Thr Gly Gln Cys Pro Cys Lys Asp Gly Val Thr Gly 420 425 430 Ile Thr Cys Asn Arg Cys Ala Lys Gly Tyr Gln Gln Ser Arg Ser Pro 435 440 445 Ile Ala Pro Cys Ile Lys Ile Pro Ala Ala Pro Pro Pro Thr Ala Ala 450 455 460 Ser Ser Thr Glu Glu Pro Ala Asp Cys Asp Ser Tyr Cys Lys Ala Ser 465 470 475 480 Lys Gly Lys Leu Lys Ile Asn Met Lys Lys Tyr Cys Lys Lys Asp Tyr 485 490 495 Ala Val Gln Ile His Ile Leu Lys Ala Glu Lys Asn Ala Asp Trp Trp 500 505 510 Lys Phe Thr Val Asn Ile Ile Ser Val Tyr Lys Gln Gly Ser Asn Arg 515 520 525 Leu Arg Arg Gly Asp Gln Thr Leu Trp Val His Ala Lys Asp Ile Ala 530 535 540 Cys Lys Cys Pro Lys Val Lys Pro Met Lys Lys Tyr Leu Leu Leu Gly 545 550 555 560 Ser Thr Glu Asp Ser Pro Asp Gln Ser Gly Ile Ile Ala Asp Lys Ser 565 570 575 Ser Leu Val Ile Gln Trp Arg Asp Thr Trp Ala Arg Arg Leu Arg Lys 580 585 590 Phe Gln Gln Arg Glu Lys Lys Gly Lys Cys Arg Lys Ala 595 600 605 581 amino acids amino acid single linear peptide not provided 2 Leu Arg Leu Leu Leu Thr Thr Ser Val Leu Arg Leu Ala Arg Ala Ala 1 5 10 15 Asn Pro Phe Val Ala Gln Gln Thr Pro Pro Asp Pro Cys Tyr Asp Glu 20 25 30 Ser Gly Ala Pro Pro Arg Cys Ile Pro Glu Phe Val Asn Ala Ala Phe 35 40 45 Gly Lys Glu Val Gln Ala Ser Ser Thr Cys Gly Lys Pro Pro Thr Arg 50 55 60 His Cys Asp Ala Ser Asp Pro Arg Arg Ala His Pro Pro Ala Tyr Leu 65 70 75 80 Thr Asp Leu Asn Thr Ala Ala Asn Met Thr Cys Trp Arg Ser Glu Thr 85 90 95 Leu His His Leu Pro His Asn Val Thr Leu Thr Leu Ser Leu Gly Lys 100 105 110 Lys Phe Glu Val Val Tyr Val Ser Leu Gln Phe Cys Ser Pro Arg Pro 115 120 125 Glu Ser Thr Ala Ile Phe Lys Ser Met Asp Tyr Gly Lys Thr Trp Val 130 135 140 Pro Tyr Gln Tyr Tyr Ser Ser Gln Cys Arg Lys Ile Tyr Gly Lys Pro 145 150 155 160 Ser Lys Ala Thr Val Thr Lys Gln Asn Glu Gln Glu Ala Leu Cys Thr 165 170 175 Asp Gly Leu Thr Asp Leu Tyr Pro Leu Thr Gly Gly Leu Ile Ala Phe 180 185 190 Ser Thr Leu Asp Gly Arg Pro Ser Ala Gln Asp Phe Asp Ser Ser Pro 195 200 205 Val Leu Gln Asp Trp Val Thr Ala Thr Asp Ile Arg Val Val Phe Ser 210 215 220 Arg Pro His Leu Phe Arg Glu Leu Gly Gly Arg Glu Ala Gly Glu Glu 225 230 235 240 Asp Gly Gly Ala Gly Ala Thr Pro Tyr Tyr Tyr Ser Val Gly Glu Leu 245 250 255 Gln Val Gly Gly Arg Cys Lys Cys Asn Gly His Ala Ser Arg Cys Val 260 265 270 Lys Asp Lys Glu Gln Lys Leu Val Cys Asp Cys Lys His Asn Thr Glu 275 280 285 Gly Pro Glu Cys Asp Arg Cys Lys Pro Phe His Tyr Asp Arg Pro Trp 290 295 300 Gln Arg Ala Ser Ala Arg Glu Ala Asn Glu Cys Leu Ala Cys Asn Cys 305 310 315 320 Asn Leu His Ala Arg Arg Cys Arg Phe Asn Met Glu Leu Tyr Lys Leu 325 330 335 Ser Gly Arg Lys Ser Gly Gly Val Cys Leu Asn Cys Arg His Asn Thr 340 345 350 Ala Gly Arg His Cys His Tyr Cys Lys Glu Gly Phe Tyr Arg Asp Leu 355 360 365 Ser Lys Ser Ile Thr Asp Arg Lys Ala Cys Lys Ala Cys Asp Cys His 370 375 380 Pro Val Gly Ala Ala Gly Lys Thr Cys Asn Gln Thr Thr Gly Gln Cys 385 390 395 400 Pro Cys Lys Asp Gly Val Thr Gly Leu Thr Cys Asn Arg Cys Ala Lys 405 410 415 Gly Phe Gln Gln Ser Arg Ser Pro Val Ala Pro Cys Ile Lys Ile Pro 420 425 430 Ala Ile Asn Pro Thr Ser Leu Val Thr Ser Thr Glu Ala Pro Ala Asp 435 440 445 Cys Asp Ser Tyr Cys Lys Pro Ala Lys Gly Asn Tyr Lys Ile Asn Met 450 455 460 Lys Lys Tyr Cys Lys Lys Asp Tyr Val Val Gln Val Asn Ile Leu Glu 465 470 475 480 Met Glu Thr Val Ala Asn Trp Ala Lys Phe Thr Ile Asn Ile Leu Ser 485 490 495 Val Tyr Lys Cys Arg Asp Glu Arg Val Lys Arg Gly Asp Asn Phe Leu 500 505 510 Trp Ile His Leu Lys Asp Leu Ser Cys Lys Cys Pro Lys Ile Gln Ile 515 520 525 Ser Lys Lys Tyr Leu Val Met Gly Ile Ser Glu Asn Ser Thr Asp Arg 530 535 540 Pro Gly Leu Met Ala Asp Lys Asn Ser Leu Val Ile Gln Trp Arg Asp 545 550 555 560 Ala Trp Thr Arg Arg Leu Arg Lys Leu Gln Arg Arg Glu Lys Lys Gly 565 570 575 Lys Cys Val Lys Pro 580 529 amino acids amino acid single linear peptide not provided 3 Tyr Cys Val Val Ser Glu Arg Gly Glu Glu Arg Val Arg Ser Cys His 1 5 10 15 Leu Cys Asn Ser Ser Asp Pro Lys Lys Ala His Pro Pro Ala Phe Leu 20 25 30 Thr Asp Leu Asn Asn Pro His Asn Leu Thr Cys Trp Gln Ser Glu Asn 35 40 45 Tyr Leu Gln Phe Pro His Asn Val Thr Leu Thr Leu Ser Leu Gly Lys 50 55 60 Lys Phe Glu Val Thr Tyr Val Ser Leu Gln Phe Cys Ser Pro Arg Pro 65 70 75 80 Glu Ser Met Ala Ile Tyr Lys Ser Met Asp Tyr Gly Arg Thr Trp Val 85 90 95 Pro Phe Gln Phe Tyr Ser Thr Gln Cys Arg Lys Met Tyr Asn Arg Pro 100 105 110 His Arg Ala Pro Ile Thr Lys Gln Asn Glu Gln Glu Ala Val Cys Thr 115 120 125 Asp Ser His Thr Asp Met Arg Pro Leu Ser Gly Gly Leu Ile Ala Phe 130 135 140 Ser Thr Leu Asp Gly Arg Pro Ser Ala His Asp Phe Asp Asn Ser Pro 145 150 155 160 Val Leu Gln Asp Trp Val Thr Ala Thr Asp Ile Arg Val Ala Phe Ser 165 170 175 Arg Leu His Thr Phe Gly Asp Glu Asn Glu Asp Asp Ser Glu Leu Ala 180 185 190 Arg Asp Ser Tyr Tyr Tyr Ala Val Ser Asp Leu Gln Val Gly Gly Arg 195 200 205 Cys Lys Cys Asn Gly His Ala Ala Arg Cys Val Arg Asp Arg Asp Asp 210 215 220 Ser Leu Val Cys Asp Cys Lys His Asn Thr Ala Gly Pro Glu Cys Asp 225 230 235 240 Arg Cys Lys Pro Phe His Tyr Asp Arg Pro Trp Gln Arg Ala Thr Ala 245 250 255 Arg Glu Ala Asn Glu Cys Val Ala Cys Asn Cys Asn Leu His Ala Arg 260 265 270 Arg Cys Arg Phe Asn Met Glu Leu Tyr Lys Leu Ser Gly Arg Lys Ser 275 280 285 Gly Gly Val Cys Leu Asn Cys Xaa Xaa Asn Thr Xaa Xaa Arg His Cys 290 295 300 His Tyr Xaa Xaa Gly Gly Xaa Leu Leu Pro Arg His Gly Lys Pro Ile 305 310 315 320 Thr His Arg Lys Ala Cys Lys Ala Cys Asp Cys His Pro Val Gly Ala 325 330 335 Ala Gly Lys Thr Cys Asn Gln Thr Thr Gly Gln Cys Pro Cys Lys Asp 340 345 350 Gly Val Thr Gly Ile Thr Cys Asn Arg Cys Ala Lys Gly Tyr Gln Gln 355 360 365 Ser Arg Ser Pro Ile Ala Pro Cys Ile Lys Ile Pro Val Arg Arg Pro 370 375 380 Thr Ala Ala Ser Xaa Val Glu Glu Xaa Xaa Glu Asp Cys Asp Ser Tyr 385 390 395 400 Cys Lys Ala Ser Lys Gly Lys Leu Lys Met Asn Met Lys Lys Tyr Cys 405 410 415 Arg Lys Asp Tyr Ala Val Gln Ile His Ile Leu Lys Ala Asp Lys Ala 420 425 430 Gly Asp Trp Trp Lys Phe Thr Val Asn Ile Ile Ser Val Tyr Lys Gln 435 440 445 Gly Thr Ser Arg Ile Arg Arg Gly Asp Gln Ser Leu Trp Ile Arg Ser 450 455 460 Arg Asp Ile Ala Cys Lys Cys Pro Lys Ile Lys Pro Leu Lys Lys Tyr 465 470 475 480 Leu Leu Leu Gly Asn Ala Xaa Asp Ser Pro Asp Gln Ser Gly Ile Val 485 490 495 Ala Asp Lys Ser Ser Leu Val Ile Gln Trp Arg Asp Thr Trp Ala Arg 500 505 510 Arg Leu Arg Lys Phe Gln Gln Arg Glu Lys Lys Gly Lys Cys Lys Lys 515 520 525 Ala 727 amino acids amino acid single linear peptide not provided 4 Met Ile Arg Gly Ile Leu Leu Leu Leu Leu Gly Thr Thr Arg Phe Ser 1 5 10 15 Pro Ile Gln Cys Ile Phe Asn Asp Val Tyr Phe Lys Met Phe Ser Gln 20 25 30 Gln Ala Pro Pro Glu Asp Pro Cys Tyr Asn Lys Ala His Glu Pro Arg 35 40 45 Ala Cys Ile Pro Asp Phe Val Asn Ala Ala Tyr Asp Ala Pro Val Val 50 55 60 Ala Ser Ser Thr Cys Gly Ser Ser Gly Ala Gln Arg Tyr Cys Glu Tyr 65 70 75 80 Gln Asp His Glu Arg Ser Cys His Thr Cys Asp Met Thr Asp Pro Leu 85 90 95 Arg Ser Phe Pro Ala Arg Ser Leu Thr Asp Leu Asn Asn Ser Asn Asn 100 105 110 Val Thr Cys Trp Arg Ser Glu Pro Val Thr Gly Ser Gly Asp Asn Val 115 120 125 Thr Leu Thr Leu Ser Leu Gly Lys Lys Phe Glu Leu Thr Tyr Val Ile 130 135 140 Leu Gln Leu Cys Pro His Ala Pro Arg Pro Asp Ser Met Val Ile Tyr 145 150 155 160 Lys Ser Thr Asp His Gly Leu Ser Trp Gln Pro Phe Gln Phe Phe Ser 165 170 175 Ser Gln Cys Arg Arg Leu Phe Gly Arg Pro Ala Arg Gln Ser Thr Gly 180 185 190 Arg His Asn Glu His Glu Ala Arg Cys Ser Asp Val Thr Arg Pro Leu 195 200 205 Val Ser Arg Ile Ala Phe Ser Thr Leu Glu Gly Arg Pro Ser Ser Arg 210 215 220 Asp Leu Asp Ser Ser Pro Val Leu Gln Asp Trp Val Thr Ala Thr Asp 225 230 235 240 Ile Arg Val Val Phe His Arg Leu Gln Arg Pro Asp Pro Gln Ala Leu 245 250 255 Leu Ser Leu Glu Ala Gly Gly Ala Thr Asp Leu Ala Ser Gly Lys Tyr 260 265 270 Ser Val Pro Leu Ala Asn Gly Pro Ala Gly Asn Asn Ile Glu Ala Asn 275 280 285 Leu Gly Gly Asp Val Ala Thr Ser Gly Ser Gly Leu His Tyr Ala Ile 290 295 300 Ser Asp Phe Ser Val Gly Gly Arg Cys Lys Cys Asn Gly His Ala Ser 305 310 315 320 Lys Cys Ser Thr Asp Ala Ser Gly Gln Leu Asn Cys Glu Cys Ser His 325 330 335 Asn Thr Ala Gly Arg Asp Cys Glu Arg Cys Lys Pro Phe His Phe Asp 340 345 350 Arg Pro Trp Ala Arg Ala Thr Ala Lys Glu Ala Asn Glu Cys Lys Glu 355 360 365 Cys Asn Cys Asn Lys His Ala Arg Gln Cys Arg Phe Asn Met Glu Ile 370 375 380 Phe Arg Leu Ser Gln Gly Val Ser Gly Gly Val Cys Gln Asn Cys Arg 385 390 395 400 His Ser Thr Thr Gly Arg Asn Cys His Gln Cys Lys Glu Gly Phe Tyr 405 410 415 Arg Asp Ala Thr Lys Pro Leu Thr His Arg Lys Val Cys Lys Ala Cys 420 425 430 Asp Cys His Pro Ile Gly Ser Ser Gly Lys Ile Cys Asn Ser Thr Ser 435 440 445 Gly Gln Cys Pro Cys Lys Asp Gly Val Thr Gly Leu Thr Cys Asn Arg 450 455 460 Cys Ala Arg Gly Tyr Gln Gln Ser Arg Ser His Ile Ala Pro Cys Ile 465 470 475 480 Lys Gln Pro Pro Arg Met Ile Asn Met Leu Asp Thr Gln Asn Thr Ala 485 490 495 Pro Glu Pro Asp Ala Pro Glu Ser Ser Pro Gly Ser Gly Gly Asp Arg 500 505 510 Asn Gly Ala Ala Glu Trp Pro Pro Ser Leu Ser Thr Ile Ala Pro Arg 515 520 525 Ala Ala Gly Val Lys Cys Gly Lys Cys Arg Val Ser Thr Lys Arg Leu 530 535 540 Asn Leu Asn Lys Phe Cys Lys Arg Asp Tyr Ala Ile Met Ala Lys Val 545 550 555 560 Ile Gly Arg Asp Thr Ser Ser Glu Ala Val Ser Arg Glu Val Gln Arg 565 570 575 Arg Ala Met Asp Pro Asp Val Ala Asp Tyr Glu Met Asp Gln Val Gln 580 585 590 Pro Gly Ser Ala Arg Ser Pro Ile Thr Gly Val Tyr Glu Phe Gln Ala 595 600 605 Ala Asp Tyr Pro Asn Pro Asn Pro Asn Pro Arg Gly Ser Glu Met Glu 610 615 620 Arg Phe Asp Leu Gln Ile Gln Ala Val Phe Lys Arg Thr Arg Pro Gly 625 630 635 640 Glu Ser Ser Gly Ala Gly Asn Val Tyr Gly Met Pro Asn Thr Thr Leu 645 650 655 Lys Arg Gly Pro Met Thr Trp Ile Ile Pro Thr Lys Asp Leu Glu Cys 660 665 670 Arg Cys Pro Arg Ile Arg Val Asn Arg Ser Tyr Leu Ile Leu Gly Arg 675 680 685 Asp Ser Glu Ala Pro Pro Gly Tyr Leu Gly Ile Gly Pro His Ser Ile 690 695 700 Val Ile Glu Trp Lys Glu Asp Trp Tyr Arg Arg Met Lys Arg Phe Gln 705 710 715 720 Arg Arg Ala Arg Thr Cys Ala 725 

What is claimed is:
 1. An isolated antibody which specifically binds a netrin consisting of the amino acid sequence of SEQ ID NO: 1, 2, 3 or 4, or portion thereof 25 residues in length, which portion modulates axon outgrowth or guidance or elicits a netrin-specific antibody.
 2. An antibody according to claim 1 which specifically binds a netrin consisting of the amino acid sequence of SEQ ID NO:1, or portion thereof 25 residues in length, which portion modulates axon outgrowth or guidance or elicits a netrin-specific antibody.
 3. An antibody according to claim 1 which specifically binds a netrin consisting of the amino acid sequence of SEQ ID NO:2 or portion thereof 25 residues in length, which portion modulates axon outgrowth or guidance or elicits a netrin-specific antibody.
 4. An antibody according, to claim 1 which specifically binds a netrin consisting of the amino acid sequence of SEQ ID NO:3, or portion thereof 25 residues in length, which portion modulates axon outgrowth or guidance or elicits a netrin-specific antibody.
 5. An antibody according to claim 1 which specifically binds a netrin consisting of the amino acid sequence of SEQ ID NO:4 or portion thereof 25 residues in length, which portion modulates axon outgrowth or guidance or elicits a netrin-specific antibody.
 6. An antibody according to claim 1 which specifically binds a netrin portion consisting of an amino acid sequence selected from the consisting of: SEQ ID NO:01, residues 289-294; SEQ ID NO:02, residues 265-270; SEQ ID NO:01, residues 296-304; SEQ ID NO:02, residues 272-280; SEQ ID NO:01, residues 308-315; SEQ ID NO:02, residues 284-291; SEQ ID NO:01, residues 320-338; SEQ ID NO:02, residues 296-319; SEQ ID NO:01, residues 345-350; SEQ ID NO:01, residues 352-368; SEQ ID NO:02, residues 328-344; SEQ ID NO:01, residues 373-380; SEQ ID NO:02, residues 349-356; SEQ ID NO:01, residues 385-401; SEQ ID NO:02, residues 361-377; SEQ ID NO:01, residues 408-416; SEQ ID NO:02, residues 384-392-392; SEQ ID NO:01, residues 418-423; SEQ ID NO:02, residues 394-399; SEQ ID NO:01, residues 427-434; SEQ ID NO:02, residues 403-410; SEQ ID NO:01, residues 439-451; SEQ ID NO:02, residues 415-427; SEQ ID NO:01, residues 453-459; SEQ ID NO:02, residues 429-435; SEQ ID NO:01, residues 466-478; SEQ ID NO:02, residues 442-454; SEQ ID NO:01, residues 485-499; SEQ ID NO:02, residues 461-475; SEQ ID NO:01, residues 513-523; SEQ ID NO:02, residues 489-499; SEQ ID NO:01, residues 545-550; SEQ ID NO:02, residues 521-526; SEQ ID NO:01, residues 573-584; SEQ ID NO:02, residues 549-560; SEQ ID NO:01, residues 528-537; SEQ ID NO:02, residues 504-513; SEQ ID NO:01, residues 40-45; SEQ ID NO:02, residues 27-30; SEQ ID NO:01, residues 51-65; SEQ ID NO:02, residues 38-52; SEQ ID NO:01, residues 68-75; SEQ ID NO:02, residues 55-62; SEQ ID NO:01, residues 97-107; SEQ ID NO:01, residues 109-116; SEQ ID NO:02, residues 80-87; SEQ ID NO:01, residues 117-123; and SEQ ID NO:02, residues 88-94.
 7. An antibody according, to claim 1 which specifically binds a netrin portion consisting of an amino acid sequence selected from the group consisting of: SEQ ID NO:01, residues 289-294; SEQ ID NO:01, residues 296-304; SEQ ID NO:01, residues 308-315; SEQ ID NO:01, residues 320-338; SEQ ID NO:01, residues 345-350; SEQ ID NO:01, residues 352-368; SEQ ID NO:01, residues 373-380; SEQ ID NO:01, residue, 385-401; SEQ ID NO:01, residues 408-416; SEQ ID NO:01, residues 418-423 ; SEQ ID NO:01, residues 427-434; SEQ ID NO:01, residues 439-451; SEQ ID NO:01, residues 453-459; SEQ ID NO:01, residues 466-478; SEQ ID NO:01, residues 485-499; SEQ ID NO:01, residues 513-523; SEQ ID NO:01, residues 545-550; SEQ ID NO:01, residues 573-584; SEQ ID NO:01, residues 528-537; SEQ ID NO:01, residues 40-45; SEQ ID NO:01, residues 51-65; SEQ ID NO:01, residues 68-75; SEQ ID NO:01, residues 97-107; SEQ ID NO:01, residues 109-116 and SEQ ID NO:01, residues 117-123.
 8. An antibody according, to claim 1 which specifically binds a netrin portion consisting of an amino acid sequence selected from the group consisting of: SEQ ID NO:02, residues 265-270; SEQ ID NO:02, residues 272-280; SEQ ID NO:02, residues 284-291; SEQ ID NO:02, residues 296-319; SEQ ID NO:02, residues 328-344; SEQ ID NO:02, residues 349-356; SEQ ID NO:02, residues 361-377; SEQ ID NO:02, residues 384-392; SEQ ID NO:02, residues 394-399; SEQ ID NO:02, residues 403-410; SEQ ID NO:02, residues 415-427; SEQ ID NO:02, residues 429-435 ; SEQ ID NO:02, residues 442-454; SEQ ID NO:02, residues 461-475; SEQ ID NO:02, residues 489-499; SEQ ID NO:02, residues 521-526; SEQ ID NO:02, residues 549-560; SEQ ID NO:02, residues 504-513; SEQ ID NO:02, residues 27-30; SEQ ID NO:residues 38-52; SEQ ID NO:02, residues 55-62; SEQ ID NO:02, residues 80-87 and SEQ ID NO:02, residues 88-94.
 9. An antibody according to claim 1 which specifically binds a netrin portion consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:01residues 289-294; SEQ ID NO:02, residues 265-270; SEQ ID NO:01, residues 296-304; SEQ ID NO:02, residues 272-280; SEQ ID NO:01, residues 308-315; SEQ ID NO:02, residues 284-291; SEQ ID NO:01, residues 320-338; SEQ ID NO:02, residues 296-319; SEQ ID NO:01, residues 345-350; SEQ ID NO:01, residues 352-368; SEQ ID NO:02, residues 328-344; SEQ ID NO:01, residues 373-380; SEQ ID NO:02, residues 349-356; SEQ ID NO:01, residues 385-401; SEQ ID NO:02, residues 361-377; SEQ ID NO:01, residues 408-416; SEQ ID NO:02, residues 384-392; SEQ ID NO:01, residues 418-423; SEQ ID NO:02, residues 394-399; SEQ ID NO:01, residues 427-434; SEQ ID NO:02, residues 403-410; SEQ ID NO:01, residues 439-451 and SEQ ID NO:02, residues 415-427.
 10. An antibody according to claim 1 which specifically binds a netrin portion consisting of an amino acid sequence selected from the group consisting of: SEQ ID NO:01, residues 453-459; SEQ ID NO:02, residues 429-435; SEQ ID NO:01, residues 466-478; SEQ ID NO:02, residues 442-454; SEQ ID NO:01, residues 485-499; SEQ ID NO:02, residues 461-475; SEQ ID NO:01, residues 513-523; SEQ ID NO:02, residues 485-499; SEQ ID NO:01, residues 545-550; SEQ ID NO:02, residues 521-526; SEQ ID NO:01, residues 573-584; SEQ ID NO:02, residues 549-560; SEQ ID NO:01, residues 528-537 and SEQ ID NO:02, residues 504-513.
 11. An antibody according to claim 1 which specifically binds a netrin portion consisting an amino acid sequence selected from the group consisting of: SEQ ID NO:01, residues 40-45; SEQ ID NO:02, residues 27-30; SEQ ID NO:01, residues 51-65; SEQ ID NO:02, residues 38-52; SEQ ID NO:01, residues 68-75; SEQ ID NO:02, residues 55-62; SEQ ID NO:01, residues 97-107; SEQ ID NO:01, residues 109-116; SEQ ID NO:02, residues 80-87; SEQ ID NO:01, residues 117-123 and SEQ ID NO:02, residues 88-94. 